Francisella tularensis, the causative agent of tularemia, is a Gram-negative facultative intracellular bacterium. Because of its extreme infectivity, high virulence, and ease of dissemination, F. tularensis is considered as a category A potential agent of bioterrorism. The virulence mechanisms of F. tularensis are poorly understood, and there are no licensed vaccines against tularemia. Our long-term goals are: (i) to understand at the molecular level the pathogenic mechanisms of F. tularensis;and (ii) identify molecular targets for effective vaccines and other therapeutics against F. tularensis infections in humans. This application is based on our recent genome-wide search for virulence determinants in F. tularensis strain LVS by signature-tagged mutagenesis (STM). This study identified 95 F. tularensis genes that are required for the in vivo survival of this pathogen. Among these are three adjacent genes (capB, capC, and capA) in a gene locus. The capBCA locus closely resembles the biosynthetic gene locus of the poly-3-glutamic acid (PGA) capsule in Bacillus anthracis, the anthrax agent. The B. anthracis capsule is a major virulence factor. However, PGA has not been described in any Gram-negative bacteria or any intracellular bacterial pathogen. Based on our exciting preliminary findings, we hypothesize that the F. tularensis capBCA locus promotes the virulence of F. tularensis by producing PGA. The Specific Aims are: (i) to characterize the F. tularensis capBCA locus, (ii) to determine PGA polymerase activity of the capBCA locus, and (iii) to determine how capB enhances F. tularensis intracellular growth and virulence. Our expertise and existing tools have provided us the unique capability to accomplish the goal of this application. We will be the first to study a potential PGA biosynthetic system in Gram-negative bacteria. The data of the proposed studies will provide valuable information concerning the significance of PGA in biology and pathogenesis of Gram-negative bacteria including F. tularensis. The PGA may be an attractive vaccine target for tularemia if it proves to be surface-exposed like its counterparts in the Gram-positive pathogens. PUBLIC HEALTH RELEVANCE: The bacterium Francisella tularensis is the causative agent of tularemia and also a bioweapon agent due to its extreme infectivity and ability to cause mortality. There are no licensed vaccines against tularemia. F. tularensis can infect a wide range of host from ticks to humans. However, the mechanisms for this extreme versatility are unknown. This project will study three bacterial genes that are potentially responsible for synthesis of an unconventional amino acid polymer called poly-glutamic acid (PGA). As the sole constituent of the capsule (coating structure) of Bacillus anthracis, causative agent of anthrax, PGA protects the bacterium from host clearance during infection of mammalian hosts. We will pursue our hypothesis that F. tularensis also produces PGA to enhance its adaptation when the bacterium is located in different microenvironments. The information will significantly enhance our knowledge in disease pathogenesis of F. tularensis. PGA can be used to develop vaccines against F. tularensis infection if it is placed at the surface of F. tularensis as its counterpart in B. anthracis